As reported in WO98/08932, protein toxins from the genus Photorhabdus have been shown to have oral toxicity against insects. The toxin complex produced by Photorhabdus luminescens (W-14), for example, has been shown to contain ten to fourteen proteins, and it is known that these are produced by expression of genes from four distinct genomic regions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotide sequences for the native toxin genes.
Of the separate toxins isolated from Photorhabdus luminescens (W-14), those designated Toxin A and Toxin B are especially potent against target insect species of interest, for example corn rootworm. Toxin A is comprised of two different subunits. The native gene tcdA (SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is an approximately 282.9 kDA protein (2516 aa) that is processed to provide TcdAii, an approximately 208.2 kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1, and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded by nucleotides 5812-7551 of SEQ ID NO:1.
Toxin B is similarly comprised of two different subunits. The native gene tcbA (SEQ ID NO:2) encodes protoxin TcbA (see SEQ ID NO:2). As determined by mass spectrometry, TcbA is processed by one or more proteases to provide Toxin B. More specifically, TcbA is an approximately 280.6 kDA (2504 aa) protein that is processed to provide TcbAii, an approximately 207.7 kDA (1844 aa) protein encoded by nucleotides 262-5793 of SEQ ID NO:2 and TcbAiii, an approximately 62.9 kDA (573 aa) protein encoded by nucleotides 5794-7512 of SEQ ID NO:2.
The native tcdA and tcbA genes are not well suited for high level expression in plants. They encode multiple destabilization sequences, mRNA splice sites, polyA addition sites and other possibly detrimental sequence motifs. In addition, the codon compositions are not like those of plant genes. WO98/08932 gives general guidance on how the toxin genes could be reengineered to more efficiently expressed in the cytoplasm of plants, and describes how plants can be transformed to incorporate the Photorhabdus toxin genes into their genomes.
In a preferred embodiment, the invention provides novel polynucleotide sequences that encode TcdA and TcbA. The novel sequences have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots, and this feature is designated by referring to the sequences as the xe2x80x9chemicotxe2x80x9d criteria, which is set forth in detail hereinafter. Other important features of the sequences are that potentially deleterious sequences have been eliminated, and unique restriction sites have been built in to enable adding or changing expression elements, organellar targeting signals, engineered protease sites and the like, if desired.
In a particularly preferred embodiment, the invention provides polynucleotide sequences that satisfy hemicot criteria and that comprise a sequence encoding an endoplasmic reticulum signal or similar targeting sequence for a cellular organelle in combination with a sequence encoding TcdA or TdbA.
More broadly, the invention provides engineered nucleic acids encoding functional Photorhabdus toxins wherein the sequences satisfy hemicot criteria.
The invention also provides transgenic plants with genomes comprising a novel sequence of the invention that imparts functional activity against insects.
SEQ ID NO:1 is the native tcdA DNA sequence together with the corresponding encoded amino acid sequence for TcdA.
SEQ ID NO:2 is the native tcbA DNA sequence together with the corresponding encoded amino acid sequence for TcbA.
SEQ ID NO:3 is an artificial sequence encoding TcdA that is suitable for expression in monocot and dicot plants.
SEQ ID NO:4 is an artificial sequence encoding TcbA that is suitable for expression in monocot and dicot plants.
SEQ ID NO:5 is an artificial hemicot sequence that encodes the 21 amino acid ER signal peptide of 15 kDa zein from Black Mexican Sweet maize.
SEQ ID NO:6 is an artificial hemicot sequence that encodes for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).
The native Photorhabdus toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus. Of particular interest are the proteins produced by the species Photorhabdus luminescens. The protein complexes have a molecular size of approximately 1,000 kDa and can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon ingestion by a number of insects.
A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabditis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (Rockville, Md.) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce insecticidal toxins. Such sources in the environment could be either terrestrial or aquatic based.
The genus Photorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits a typical of this family. For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and bioluminescent. This latter trait is otherwise unknown within the Enterobacteriaceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on DNAxe2x80x94DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus) or absence (Xenorhabdus) of catalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Photorhabdus; Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Photorhabdus from Xenorhabdus.
Currently, the bacterial genus Photorhabdus is comprised of a single defined species, Photorhabdus luminescens (ATCC Type strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A variety of related strains have been described in the literature (e.g., Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56, 181-186).
The following toxin producing Photorhabdus strains have been deposited:
All strains were deposited in accordance with the terms of the Budapest Treaty. Strains having accession numbers prefaced by xe2x80x9cATTCxe2x80x9d were deposited on the indicated date in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA. Strains prefaced by xe2x80x9cNRRLxe2x80x9d were deposited on the indicated date in the Agricultural Research Service Patent Culture Collection (NRRL), National Center for Agricultural Utilization Research, ARS-USDA, 1815 North University St., Peoria Ill. 61604 USA.
The present invention provides hemicot nucleic acid sequences encoding toxins from any Photorhabdus species or strain that produces a toxin having functional activity. Hemicot nucleic acid sequences encoding proteins homologous to such toxins are also encompassed by the invention.
Several terms that are used herein have a particular meaning and are defined as follows:
By xe2x80x9cfunctional activityxe2x80x9d it is meant herein that the protein toxins) function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect. When an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression, formulated protein compositions), sprayable protein compositions), a bait matrix or other delivery system, the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.
By xe2x80x9chomologxe2x80x9d it is meant an amino acid sequence that is identified as possessing homology to a reference Photorhabdus toxin polypeptide amino acid sequence.
By xe2x80x9chomologyxe2x80x9d it is meant an amino acid sequence that has a similarity index of at least 33% and/or an identity index of at least 26% to a reference Photorhabdus toxin polypeptide amino acid sequence, as scored by the GAP algorithm using the B10sum 62 protein scoring matrix Wisconsin Package Version 9.0, Genetics Computer Group GCG), Madison, Wis.).
By xe2x80x9cidentityxe2x80x9d is meant an amino acid sequence that contains an identical residue at a given position, following alignment with a reference Photrhabdus toxin polypeptide amino acid sequence by the GAP algorithm.
By the use of the term xe2x80x9cPhotorhabdus toxinxe2x80x9d it is meant any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via baculovirus, or delivered by any other applicable host or delivery system.
By the use of the term xe2x80x9ctoxicxe2x80x9d or xe2x80x9ctoxicityxe2x80x9d as used herein it is meant that the toxins produced by Photorhabdus have xe2x80x9cfunctional activityxe2x80x9d as defined herein.
By xe2x80x9csubstantial sequence homologyxe2x80x9d is meant either: a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment to produce a protein having similar biochemical properties; or a polypeptide having an amino acid sequence sufficiently similar to another polypeptide to exhibit similar biochemical properties.
As with other bacterial toxins, the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist. Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein. Preferably, the toxins are active against Lepidoptera, Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina. The inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative strains thereof, as well as any protein toxins produced by Photorhabdus. These homologous proteins may differ in sequence, but do not differ in function from those toxins described herein. Homologous toxins are meant to include protein complexes of between 300 kDa to 2,000 kDa and are comprised of at least two 2) subunits, where a subunit is a peptide which may or may not be the same as the other subunit. Various protein subunits have been identified and are taught in the Examples herein. Typically, the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.
As discussed above, some Photorhabdus strains can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the phylum Nematoda, have evolved an ability to exploit insect larvae as a favored growth environment. The insect larvae provide a source of food for growing nematodes and an environment in which to reproduce. One dramatic effect that follows invasion of larvae by certain nematodes is larval death. Larval death results from the presence of, in certain nematodes, bacteria that produce an insecticidal toxin which arrests larval growth and inhibits feeding activity.
Interestingly, it appears that each genus of insect parasitic nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode. In the interim since this research was initiated, the name of the bacterial genus Xenorhabdus was reclassified into the Xenorhabdus and the Photorhabdus. Bacteria of the genus Photorhabdus are characterized as being symbionts of Heterorhabditus nematodes while Xenorhabdus species are symbionts of the Steinernema species. This change in nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the inventions described herein.
The peptides and genes that are disclosed herein are named according to the guidelines recently published in the Journal of Bacteriology xe2x80x9cInstructions to Authorsxe2x80x9d p. i-xii January 1996), which is incorporated herein by reference.
Transformation methods useful in carrying out the invention are well known, and are described, for example, in WO98/08932.
Hemicot tcdA and tcbA
SEQ ID NO: 3 is the nucleotide sequence for an engineered tcdA gene in accordance with the invention. SEQ ID NO: 4 is the nucleotide sequence for an engineered tcbA gene in accordance with the invention.
The following Tables 1 and 2 identify significant features of the engineered tcdA and tcbA genes.
It should be noted that the proteins encoded by the plant-optimized tcdA (SEQ ID NO:3) and tcbA (SEQ ID NO:5) differ from the native proteins by the addition of an Ala residue at position #2. This modification was made to accommodate the NcoI site which spans the ATG start codon.
The following Table 3 compares the codon composition of the engineered tcdA gene of SEQ ID NO:3 and engineered tcbA gene of SEQ ID NO:5 with the codon compositions of the native genes, the typical dicot genes, and maize genes.